Title

Author

Date of Award

Level of Access

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

John Pierce Wise, Sr.

Second Committee Member

Greg Mayer

Third Committee Member

Stephen C. Pelsue

Abstract

Particulate hexavalent chromium (Cr(VI)) compounds are human lung carcinogens. However, their carcinogenicity is poorly understood. The best model for Cr(VI)-induced carcinogenesis involves the acquisition of structural and numerical chromosome instability (CIN). Many mechanisms contribute to CIN. Among these, centrosomes play a pivotal role because they dictate proper segregation of chromosomes during cell division. Cr(VI) causes centrosome amplification, a phenotype where cells have extra centrosomes and hence can undergo unequal distribution of chromosomes resulting in CIN. How Cr(VI) induces these abnormalities is unknown. Moreover, whether Cr(VI)-induced centrosome amplification is a permanent phenotypic change is also unknown. This work investigates the permanence of the centrosome amplification phenotype and explores centrosome defects that can lead to centrosome amplification.

We analyzed centrosome numbers in clonal cell lines developed from repeated exposures to Cr(VI). We found that these cells lines develop a permanent centrosome amplification phenotype after one, two or three exposure to Cr(VI). Moreover, permanent centrosome amplification correlates with the acquisition of permanent changes in chromosome numbers and hence, numerical CIN.

In addition, we investigated centriole disengagement which is the licensing step of centrosome duplication. We found that chronic exposure to particulate Cr(VI) induces premature centriole disengagement in interphase. Consistent with this, we observed a decrease in securin and increase in active separase and Plk1, which regulate the disruption of the S-M protein linker that holds centrioles together.

Chronic exposure to Cr(VI) also caused premature centrosome separation in interphase, suggesting that Cr(VI) has the potential to affect the other centrosome protein linker (i.e. G1-G2 tether) which holds duplicated centrosomes together. Cr(VI) also decreases protein levels and localization of Nek2 and Eg5, proteins involved in centrosome separation.

Overall, the data indicate that Cr(VI) targets many aspects of centrosome biology. We propose that disruption of the G1-G2 tether leads to premature disruption of the S-M linker causing centriole disengagement and triggering centrosome duplication. This causes centrosome amplification, which leads to numerical CIN and ultimately, neoplastic transformation and cancer.